C O M M U N I C A T I O N S
Figure 1. Sequence-specific two-step labeling of plasmid DNA. (a) pBR322 DNA was amino-modified with M.HhaI or M.TaqI in the presence of cofactor
2 and then treated with a cyanine-5 N-hydroxysuccinimide ester (Cy5-NHS). Labeled DNA was fragmented with R.GsuI endonuclease to produce fragments
F1-F4 and analyzed by agarose gel electrophoresis. Imaging of the Cy5 fluorescence was performed using a 635 nm laser scanner. A DNA fragment (F3)
containing no M.TaqI sequences is not visible in the scan. Part C shows unmodified pBR322 DNA treated with Cy5-NHS followed by R.GsuI fragmentation.
(b) pBR322 DNA was amino-modified with M.TaqI in the presence of cofactor 2 and then treated with biotin-NHS. Labeled DNA was fragmented with
R.FspAI and R.MbiI endonucleases to produce fragments F1′-F4′. The left panel shows an analysis by agarose gel electrophoresis of all DNA fragments
(T ) total), fragments not retained on streptavidin-coated magnetic beads (F ) flow through), and fragments recovered from streptavidin-coated magnetic
beads by extraction with phenol/chloroform (B ) bound). The right panel shows the same analysis except that pBR322 DNA premethylated with M.BseCI
and AdoMet 1 was used. The changed distribution of fragments shows that M.TaqI-specific biotinylation is blocked at the premethylated M.BseCI site.
amino-functionalized with M.TaqI and treated with NHS-biotin.
M.BseCI methylates the second adenine residue within the hexa-
nucleotide 5′-ATCGAT-3′ sequence, which overlaps with one of
the seven M.TaqI sites in pBR322. Both plasmids were cleaved
with the R.FspAI and R.MbiI endonucleases, and the fragments
were passed over streptavidin beads to selectively capture biotin-
containing fragments (Figure 1b). Indeed our experiment shows
that quantitative M.TaqI-specific biotinylation is achieved. Most
importantly, a different distribution pattern is observed with the
M.BseCI-premethylated plasmid: the shortest fragment (F4′) is no
longer retained on streptavidin-coated beads but is detected in the
flow-through fraction. Clearly, enzymatic methylation by M.BseCI
blocks the single M.TaqI site on this fragment for amino-
functionalization by M.TaqI and subsequent biotinylation, and
therefore methylated and unmethylated target sites can be quanti-
tatively discriminated. This result demonstrates the utility of mTAG
labeling to query the methylation status of specific sequences in
DNA, paving the way to novel approaches for methylation profiling
of genomic DNA.
sequences, offering unprecedented experimental control over
sequence-specific manipulation of DNA. This new class of mo-
lecular tools envisions many potential applications9 ranging from
probes for genetic screening to molecular building blocks in DNA-
based nanobiotechnology.
Acknowledgment. The authors thank M. Krenevicˇiene˘ for
recording NMR spectra, J. Sˇlyzˇiujte˘ for assistance with preparing
M.HhaI mutants, and Prof. M. Kokkinidis for a gift of M.BseCI.
This work was supported by grants from the Internationale Bu¨ro
des BMBF, the Ministry of Education and Science of Lithuania,
and the Lithuanian State Science and Study Foundation.
Supporting Information Available: Supporting figures, methods
for the synthesis of AdoMet analogue 2, DNA labeling, and analysis
of labeled DNA. This material is available free of charge via the Internet
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